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Development of cell-active BRD4-D1 selective inhibitors to decode the role of BET proteins in LPS-mediated liver
Cole R Scholtz1, Luke C Doskey2, Sai Giridhar Sarma Kandanur1
1Department of Chemistry, University of Minnesota, Minneapolis, MN, 55455, United States.
Abstract:
The endogenously expressed BET proteins (BRD2, BRD3, BRD4) are upstream clinical targets for anti-inflammatory treatments, where inhibition of the tandem bromodomains (D1 and D2) have proven efficacious in vitro and in vivo towards NF-κB-mediated inflammation. Despite their efficacy, dose-limiting toxicities associated with BET inhibition have limited clinical progression. One strategy to circumvent these dose-limiting toxicities has included domain- or protein-selective inhibition of the BET bromodomains. Based on previously reported 1,2,4-substituted imidazole scaffolds, we characterize and report on next-generation BRD4-D1 selective inhibitors, 39 and 41. Compound 39 is both highly potent and selective towards BRD4-D1 (Ki = 2.9 ± 1.0 nM, >1700-fold over BRD2-D1 via fluorescence anisotropy) over other BET bromodomains in addition to being cell-active at nanomolar concentrations. We also characterized 39's solubility and cellular activity in addition to its off-target hERG liability (a common cardiovascular risk for drug candidates). An acetylated analogue, 41, had an 80-fold reduced hERG affinity compared to previous BRD4-D1 selective compounds. In the context of liver inflammation, we screened 39 against an LPS-mediated cellular model of liver inflammation. Upon treatment with 39, pro-inflammatory chemokines CXCL1 and CCL2 transcripts were significantly downregulated compared to the control; however, BRD4-D1 selective inhibition remained insufficient to reproduce the anti-inflammatory activity of pan-BET treatment. On a mechanistic level, these data highlight that more than one bromodomain within the BET family may be contributing to CXCL1 and CCL2 expression, where multi-domain inhibition or other therapeutic modalities may be needed in these contexts to achieve sufficient anti-inflammatory effects.
Insights
Researchers developed novel BRD4-D1 selective inhibitors for anti-inflammatory treatments. While potent, BRD4-D1 inhibition alone was insufficient for liver inflammation, suggesting multi-domain BET inhibition is needed.
Area of Science:
- Medicinal Chemistry
- Pharmacology
- Inflammation Research
Background:
- BET proteins (BRD2, BRD3, BRD4) are targets for anti-inflammatory therapies.
- Inhibiting BET bromodomains shows efficacy but faces dose-limiting toxicities.
- Domain- or protein-selective inhibition is a strategy to mitigate toxicity.
Purpose of the Study:
- To develop next-generation BRD4-D1 selective inhibitors.
- To characterize novel compounds for potency, selectivity, and cellular activity.
- To evaluate BRD4-D1 selective inhibition in a liver inflammation model.
Main Methods:
- Design and synthesis of 1,2,4-substituted imidazole scaffolds.
- Biochemical assays (fluorescence anisotropy) for BET bromodomain inhibition.
- Cell-based assays for inflammation (LPS model) and off-target liability (hERG).
Main Results:
- Compound 39 demonstrated high potency and selectivity for BRD4-D1.
- Compound 39 was cell-active and showed reduced hERG liability compared to prior compounds.
- BRD4-D1 selective inhibition partially reduced inflammatory markers but was less effective than pan-BET inhibition in liver inflammation.
Conclusions:
- Novel BRD4-D1 selective inhibitors were successfully developed.
- BRD4-D1 inhibition alone may not be sufficient for certain inflammatory conditions.
- Multi-domain BET inhibition or alternative strategies may be required for robust anti-inflammatory effects.
